Abstract Background: Infiltration of immunosuppressive tumor-associated macrophages (TAMs) after radiation therapy (RT) contributes to poor outcomes in breast cancer. Although pharmacologic and antibody-based TAM-targeting strategies have been explored, they have shown limited clinical success. We hypothesized that genetically reprogramming TAMs by removing a central regulator of their immunosuppressive phenotype would yield more durable functional remodeling. In this study, we identify the transcription factor GATA3 as a key driver of pro-tumor TAM activity. Methods: We utilized an orthotopic, syngeneic murine model of triple-negative breast cancer in mice with or without myeloid/macrophage-specific ablation of GATA3 (mG3KO). Following RT, we performed single-cell RNA sequencing (scRNA-seq) on tumor-infiltrating lymphocytes. scRNA-seq findings were then correlated with patient survival by analyzing TCGA breast cancer (TCGA-BRCA) and pan-cancer (TCGA-PANCAN) datasets. TCGA-BRCA samples were deconvolved using BayesPrism, while published immune cell fractions were used for TCGA-PANCAN. Samples with inferred macrophage fractions 3% were retained in both datasets, and gene set scoring was performed using GSVA. Samples were stratified based on GSVA scores for survival analysis, and associations were tested using the log-rank test and Cox proportional-hazards regressions. Results: scRNA-seq revealed that GATA3 ablation markedly reduced immunosuppressive, immune-regulatory TAM populations (Arg1, Hmox1, Hilpda, Spp1) with a corresponding increase in inflammatory lipid-associated TAMs (Apoe, Acp5, C1qa, C1qb). Differential gene expression analysis between mG3KO and control TAMs identified a GATA3 program (G3P) enriched for immunosuppressive genes and chemotactic factors (Arg1, Hmox1, Hilpda, Spp1, Inhba, Cd274, Cxcl2, Cxcl3, Ccl3). In contrast, genes negatively regulated by GATA3 (G3NP) were linked to lipid metabolism and antigen presentation (Adgre1, Apoe, Lpl, Acp5, H2-Aa, H2-Eb1, Cd74). In TCGA-BRCA, enrichment for G3P in macrophages correlated with significantly worse overall survival, while elevated G3NP expression predicted improved survival. Similarly, in TCGA-PANCAN, both overall survival and progression-free interval were poorer in G3Phi tumors, with opposite trends observed in G3NPhi tumors. Conclusion: GATA3 is a previously unrecognized, central regulator of TAM immunosuppressive programming. Its deletion in myeloid cells reprograms TAMs toward a pro-inflammatory, antitumor state, and GATA3-dependent gene signatures strongly associate with clinical outcomes across multiple human cancers. These findings highlight GATA3 as a compelling target for macrophage-based genetic engineering and targeting strategies to enhance radiotherapy efficacy. Citation Format: Harry Xiao, Tahir B. Dar, Satchel Stevens, Jolene Viramontes, Stephen L. Shiao. Single-cell and bulk transcriptomics uncovers GATA3-mediated macrophage immunosuppressive programming in murine and human cancers abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A043.
Xiao et al. (Wed,) studied this question.
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